Protocols

Fish genomic DNA preparation

Useful as a quick easy method for large amounts of good quality DNA from fish blood. Can be scaled down but as fish red cells are nucleated be sure to to get a sufficiently small amount of blood for a miniprep.

Take 100-300µl of fish blood and add to 10ml of 50mM Tris/HCl, pH 8.5, 20mM EDTA, 200mM NaCl, 0.01% SDS, 20µg RNAaseA in a 50ml polypropylene Universal. Gently mix (eg rotary or rolling on a shaking platform) for 15mins. Add 10ml of phenol, pH 8.0 and gently mix for 1 hour (or more) until emulsified.
Spin 3000g for 10min and remove supernatant with truncated blue tip or 10ml pippette to a fresh tube. The supernatant will be viscous, stringy and cloudy and will be difficult to separate from the interphase crud. Dont worry too much just do your best.
Add 5ml of 50mM Tris/HCl, pH 8.5, 20mM EDTA, 200mM NaCl, 0.01% SDS and mix gently. The stringy cloudy solution should go clear. Extract with an equal volume of chloroform for 1 hour as before. Spin again and collect 10ml of supernatant as before. Be more careful this time- avoid the interphase.
Add 2 volumes of ethanol and invert several times. Pick up the high molecular weight DNA on a bent pippete tip.
Slide DNA of the tip into a 1.5ml microtube containing 1ml of 70% ethanol and leave for for 5 min. Spin for 1min at full speed in microfuge, decant supernatant, pulse spin down the excess liquid and remove with a pippete. Leave to dry for 5min (ie lid open on the bench). Add 0.5 to 1ml of sterile distilled water (or TE). Leave in the fridge overnight to rehydrate, gently mix next day and store as required.
Determine the DNA concentration spectrophotometrically and store at -20°C to 4°C. This DNA is usually high Mwt and good for all purposes.

Notes - Do not use more than 300µl of blood in this protocol. Scale up if required. 300µl of fish blood should yield at least 0.5mg of DNA. Contamination can be a problem in Southern blot experiments where large amounts of plasmids are being routinely used as very small amounts carried over from, for example, a contaminated tip can overwhelm a Southern blot. Therefore all reagents should be made up from scratch, autoclaved and marked GENOMIC DNA ONLY and only fresh tips should be used. The same points should be noted when restriction digesting and running the DNA ie ideally seperate enzymes and buffers should be kept for this purpose.